Hybrid cell line for producing monoclonal antibody to human T cells

ABSTRACT

Hybrid cell line for production of monoclonal antibody to an antigen found on all normal human T cells. The hybrid is formed by fusing splenocytes from immunized Balb/cJ mice with P3X63Ag8U1 myeloma cells. Diagnostic and therapeutic uses of the monoclonal antibody are also disclosed.

This is a division of application Ser. No. 22,132, filed Mar. 20, 1979,now U.S. Pat. No. 4,363,799.

FIELD OF THE INVENTION

This invention relates generally to new hybrid cell lines and morespecifically to hybrid cell lines for production of monoclonal antibodyto an antigen found on all normal human T cells, to the antibody soproduced, and to therapeutic and diagnostic methods employing thisantibody.

DESCRIPTION OF THE PRIOR ART

The fusion of mouse myeloma cells to sploen cells from immunized mice byKohler and Milstein in 1975 [Nature 256, 495-497 (1975)] demonstratedfor the first time that it was possible to obtain a continuous cell linemaking homogeneous (so-called "monoclonal") antibody. Since this seminalwork, much effort has been directed to the production of various hybridcells (called "hybridomas") and to the use of the antibody made by thesehybridomas for various scientific investigations. See, for example,Current Topics in Microbiology and Immunology, Volume 81--"LymphocyteHybridomas", F. Melchers, M. Potter, and N. Warner, Editors,Springer-Verlag, 1978, and references contained therein; C. J.Barnstable, et al., Cell, 14, 9-20 (May, 1978); P. Parham and W. F.Bodmer, Nature 276, 397-399 (November, 1978); Handbook of ExperimentalImmunology, Third Edition, Volume 2, D. M. Wier, Editor, Blackwell,1978, Chapter 25; and Chemical and Engineering News, Jan. 1, 1979,15-17.

These references simultaneously indicates the rewards and complicationsof attempting to produce monoclonal antibody from hybridomas. While thegeneral technique is well understood conceptually, there are manydifficulties met and variations required for each specific case. Infact, there is no assurance, prior to attempting to prepare a givenhybridoma, that the desired hybridoma will be obtained, that it willproduce antibody if obtained, or that the antibody so produced will havethe desired specificity. The degree of success is influenced principallyby the type of antigen employed and the selection technique used forisolating the desired hybridoma.

The attempted production of monoclonal antibody to human lymphocytecell-surface antigens has been reported only in a few instances. See,for example, Current Topics in Microbiology and Immunology, ibid, 66-69and 164-169. The antigens used in these reported experiments werecultured human lymphoblastoid leukemia and human chronic lymphocyticleukemia cell lines. Many hybridomas obtained appeared to produceantibody to various antigens on all human cells. None of the hybridomasproduced antibody against a predefined class of human lymphocytes.

It should be understood that there are two principal classes oflymphocytes involved in the immune system of humans and animals. Thefirst of these (the thymus-derived cell or T cell) is differentiated inthe thymus from haemopoietic stem cells. While within the thymus, thedifferentiating cells are termed "thymocytes." The mature T cells emergefrom the thymus and circulate between the tissues, lymphatics, and thebloodstream. These T cells form a large proportion of the pool ofrecirculating small lymphocytes. They have immunological specificity andare directly involved in cell-mediated immune responses (such as graftrejection) as effector cells. Although T cells do not secrete humoralantibodies, they are sometimes required for the secretion of theseantibodies by the second class of lymphocytes discussed below. Sometypes of T cells play a regulating function in other aspects of theimmune system. The mechanism of this process of cell cooperation is notyet completely understood.

The second class of lymphocytes (the bond marrow-derived cells or Bcells) are those which secrete antibody. They also develop fromhaemopoietic stem cells, but their differentiation is not determined bythe thymus. In birds, they are differentiated in an organ analogous tothe thymus, called the Bursa of Fabricius. In mammals, however, noequivalent organ has been discovered, and it is thought that these Bcells differentiate within the bone marrow.

It is now recognized that T cells are divided into at least severalsubtypes, termed "helper", "suppressor", and "killer" T cells, whichhave the function of (respectively) promoting a reaction, suppressing areaction, or killing (lysing) foreign cells. These subclasses are wellunderstood for murine systems, but they have only recently beendescribed for human systems. See, for example, R. L. Evans, et al.,Journal of Experimental Medicine, Volume 145, 221-232, 1977; and L.Chess and S. F. Schlossman--"Functional Analysis of Distinct HumanT-Cell Subsets Bearing Unique Differentiation Antigens", in"Contemporary Topics in Immunobiology", O. Stutman, Editor, PlenumPress, 1977, Volume 7, 363-379.

The ability to identify or suppress classes or subclasses of T cells isimportant for diagnosis or treatment of various immunoregulatorydisorders or conditions.

For example, certain leukemias and lymphomas have differing prognosisdepending on whether they are of B cell or T cell origin. Thus,evaluation of the disease prognosis depends upon distinguishing betweenthese two classes of lymphocytes. See, for example, A. C. Aisenberg andJ. C. Long, The American Journal of Medicine, 58:300 (March, 1975); D.Belpomme, et al., in "Immunological Diagnosis of Leukemias andLymphomas", S. Thierfelder, et al., eds, Springer, Heidelberg, 1977,33-45; and D. Belpomme, et al., British Journal of Haematology, 1978,38, 85. Certain disease states (e.g., juvenile rheumatoid arthritis andcertain leukemias) are associated with an imbalance of T cellsubclasses. It has been suggested that autoimmune diseases generally areassociated with an excess of "helper" T cells or a deficiency of certain"suppressor" T cells, while malignancies generally are associated withan excess of "suppressor" T cells. In certain leukemias, excess T cellsare produced in an arrested stage of development. Diagnosis may thusdepend on the ability to detect this imbalance or excess. See, forexample, J. Kersey, et al., "Surface Markers Define Human LymphoidMalignancies with Differing Prognoses" in Haematology and BloodTransfusion, Volume 20, Springer-Verlag, 1977, 17-24, and referencescontained therein.

On the therapeutic side, there is some suggestion, as yet not definitelyproven, that administration of antibodies against the subtype of T cellin excess may have therapeutic benefit in autoimmune disease ormalignancies. Antisera against the entire class of human T cells(so-called antihuman thymocyte globulin or ATG) has been reported usefultherapeutically in patients receiving organ transplants. Since thecell-mediated immune response (the mechanism whereby transplants arerejected) depends upon T cells, administration of antibody to T cellsprevents or retards this rejection process. See, for example, Cosimi, etal., "Randomized Clinical Trial of ATG in Cadaver Renal AllgraftRecipients: Importance of T Cell Monitoring", Surgery 40:155-163 (1976)and references contained therein.

The identification and suppression of human T cell classes andsubclasses has previously been accomplished by the use of spontaneousantoantibodies or selective antisera for human T cells obtained byimmunizing animals with human T cells, bleeding the animals to obtainserum, and adsorbing the antiserum with (for example) autologous but notallogeneic B cells to remove antibodies with unwanted reactivities. Thepreparation of these antisera is extremely difficult, particularly inthe adsorption and purification steps. Even the adsorbed and purifiedantisera contain many impurities in addition to the desired antibody,for several reasons. First, the serum contains millions of antibodymolecules even before the T cell immunization. Second, the immunizationcauses production of antibodies against a variety of antigens found onall human T cells injected. There is no selective production of antibodyagainst a single antigen. Third, the titer of specific antibody obtainedby such methods is usually quite low, (e.g., inactive at dilutionsgreater than 1:100) and the ratio of specific to non-specific antibodyis less than 1/10⁶.

See, for example, the Chess and Schlossman article referred to above (atpages 365 and following) and the Chemical and Engineering News articlereferred to above, where the deficiencies of prior art antisera and theadvantages of monoclonal antibody are described.

SUMMARY OF THE INVENTION

There has now been discovered a novel hybridoma which is capable ofproducing novel monoclonal antibody against an antigen found onessentially all normal human peripheral T cells. The antibody soproduced is monospecific for a single determinant on normal human Tcells and contains essentially no other anti-human immuneglobulin, incontrast to prior art antisera (which are inherently contaminated withantibody reactive to numerous human antigens) and to prior artmonoclonal antibodies (which are not monospecific for a human T cellantigen). Moreover, this hybridoma can be cultured to produce antibodywithout the necessity of immunizing and killing animals, followed by thetedious adsorption and purification steps necessary to obtain even theimpure antisera of the prior art.

It is accordingly one object of this invention to provide hybridomaswhich produce antibodies against an antigen found on essentially allnormal human T cells.

It is a further aspect of the present invention to provide methods forpreparing these hybridomas.

A further object of the invention is to provide essentially homogeneousantibody against an antigen found on essentially all normal human Tcells.

A still further object is to provide methods for treatment or diagnosisof disease employing these antibodies.

Other objects and advantages of the invention will become apparent fromthe examination of the present disclosure.

In satisfaction of the foregoing objects and advantages, there isprovided by this invention a novel hybridoma producing novel antibody toan antigen found on essentially all normal human T cells, the antibodyitself, and diagnostic and therapeutic methods employing the antibody.The hybridoma was prepared generally following the method of Milsteinand Kohler. Following immunization of mice with normal E rosettepositive human T cells, the spleen cells of the immunized mice werefused with cells from a mouse myeloma line and the resultant hybridomasscreened for those with supernatants containing antibody which gaveselective binding to normal E rosette positive human T cells. Thedesired hybridomas were subsequently cloned and characterized. As aresult, a hybridoma was obtained which produces antibody (designatedOKT1) against an antigen on essentially all normal human T cells. Notonly does this antibody react with essentially all normal humanperipheral T cells, but it also does not react with other normalperipheral blood lymphoid cells. In addition, the cell surface antigenrecognized by this antibody is detected on only mature thymocytes and iscompletely lacking on greater than 90% of normal human thymocytes.

In view of the difficulties indicated in the prior art and the lack ofsuccess reported using malignant cell lines as the antigen, it wassurprising that the present method provided the desired hybridoma. Itshould be emphasized that the unpredictable nature of hybrid cellpreparation does not allow one to extrapolate from one antigen or cellsystem to another. In fact, the present applicants have discovered thatusing a T cell malignant cell line as the antigen caused formation ofhybridomas which did not produce the desired antibody. Attempts to usepurified antigens separated from the cell surfaces were alsounsuccessful.

The preparation and characterization of the hybridoma and the resultantantibody will be better understood by reference to the followingdescription and Examples.

DETAILED DESCRIPTION OF THE INVENTION

The method of preparing the hybridoma generally comprises the followingsteps:

A. Immunizing mice with E rosette positive purified normal humanperipheral T cells. While it has been found that female Balb/cJ mice arepreferred, it is contemplated that other mouse strains could be used.The immunization schedule and T cell concentration should be such as toproduce useful quantities of suitably primed splenocytes. Threeimmunizations at fourteen day intervals with 2×10⁷ cells/mouse/injectionin 0.2 ml phosphate buffered saline has been found to be effective.

B. Removing the spleens from the immunized mice and making a spleensuspension in an appropriate medium. About one ml of medium per spleenis sufficient. These experimental techniques are well-known.

C. Fusing the suspended spleen cells with mouse myeloma cells from asuitable cell line by the use of a suitable fusion promoter. Thepreferred ratio is about 5 spleen cells per myeloma cell. A total volumeof about 0.5-1.0 ml of fusion medium is appropriate for about 10⁸splenocytes. Many mouse myeloma cell lines are known and available,generally from members of the academic community or various depositbanks, such as the Salk Institute Cell Distribution Center, La Jolla,CA. The cell line used should preferably be of the so-called "drugresistant" type, so that unfused myeloma cells will not survive in aselective medium, while hybrids will survive. The most common class is8-azaguanine resistant cell lines, which lack the enzyme hypoxanthineguanine phophoribosyl transferase and hence will not be supported by HAT(hypoxanthine, aminopterin, and thymidine) medium. It is also generallypreferred that the myeloma cell line used be of the so-called"non-secreting" type, in that it does not itself produce any antibody,although secreting types may be used. In certain cases, however,secreting myeloma lines may be preferred. While the preferred fusionpromoter is polyethylene glycol having an average molecular weight fromabout 1000 to about 4000 (commercially available as PEG 1000, etc.),other fusion promoters known in the art may be employed.

D. Diluting and culturing in separate containers, the mixture of unfusedspleen cells, unfused myeloma cells, and fused cells in a selectivemedium which wll not support the unfused myeloma cells for a timesufficient to allow death of the unfused cells (about one week). Thedilution may be a type of limiting one, in which the volume of diluentis statistically calculated to isolate a certain number of cells (e.g.,1-4) in each separate container (e.g., each well of a microtiter plate).The medium is one (e.g., HAT medium) which will not support thedrug-resistant (e.g., 8-azaguanine resistant) unfused myeloma cell line.Hence, these myeloma cells perish. Since the unfused spleen cells arenon-malignant, they have only a finite number of generations. Thus,after a certain period of time (about one week) these unfused spleencells fail to reproduce. The fused cells, on the other hand, continue toreproduce because they possess the malignant quality of the myelomaparent and the ability to survive in the selective medium of the spleencell parent.

E. Evaluating the supernatant in each container (well) containing ahybridoma for the presence of antibody to E rosette positive purifiedhuman T cells.

F. Selecting (e.g., by limiting dilution) and cloning hybridomasproducing the desired antibody.

Once the desired hybridoma has been selected and cloned, the resultantantibody may be produced in one of two ways. The purest monoclonalantibody is produced by in vitro culturing of the desired hybridoma in asuitable medium for a suitable length of time, followed by recovery ofthe desired antibody from the supernatant. The suitable medium andsuitable length of culturing time are known or are readily determined.This in vitro technique produces essentially monospecific monoclonalantibody, essentially free from other specific antihuman immuneglobulin. There is a small amount of other immune globulin present sincethe medium contains xenogenic serum (e.g., fetal calf serum). However,this in vitro method may not produce a sufficient quantity orconcentration of antibody for some purposes, since the concentration ofmonoclonal antibody is only about 50 μg/ml.

To produce a much greater concentration of slightly less pure monoclonalantibody, the desired hybridoma may be injected into mice, preferablysyngenic or semisyngenic mice. The hybridoma will cause formation ofantibody-producing tumors after a suitable incubation time, which willresult in a high concentration of the desired antibody (about 5-20mg/ml) in the bloodstream and peritoneal exudate (ascites) of the hostmouse. Although these host mice also have normal antibodies in theirblood and ascites, the concentration of these normal antibodies is onlyabout 5% of the monoclonal antibody concentration. Moreover, since thesenormal antibodies are not antihuman in their specificity, the monoclonalantibody obtained from the harvested ascites or from the serum isessentially free of any contaminating antihuman immune globulin. Thismonoclonal antibody is high titer (active at dilutions of 1:30,000 orhigher) and high ratio of specific to non-specific immune globulin(about 1/20). Immune globulin produced incorporating the κ light myelomachains are non-specific, "nonsense" peptides which merely dilute themonoclonal antibody without detracting from its specificity.

EXAMPLE I Production of Monoclonal Antibodies A. Immunization andSomatic Cell Hybridization

Female Balb/cJ mice (Jackson Laboratories; 6-8 weeks old) were immunizedintraperitoneally with 2×10⁷ E rosette purified T cells in 0.2 ml ofphosphate buffered saline at 14-day intervals. Four days after the thirdimmunization, spleens were removed from the mice, and a single cellsuspension was made by pressing the tissue through a stainless steelmesh.

Cell fusion was carried out according to the procedure developed byKohler and Milstein. 1×10⁸ splenocytes were fused in 0.5 ml of a fusionmedium comprising 35% polyethylene glycol (PEG 1000) and 5%dimethylsulfoxide in RPMI 1640 medium (Gibco, Grand Island, N.Y.) with2×10⁷ P3X63Ag8U1 myeloma cells supplied by Dr. M. Scharff, AlbertEinstein College of Medicine, Bronx, NY. These myeloma cells secreteIgG₁ κ light chains.

B. Selection and Growth of Hybridoma

After cell fusion, cells were cultured in HAT medium (hypoxanthine,aminopterin, and thymidine) at 37° C. with 5% CO₂ in a humid atmosphere.Several weeks later, 40 to 100 μl of supernatant from culturescontaining hybridomas were added to a pellet of 10⁶ peripherallymphocytes separated into E rosette positive (E⁺) and E rosettenegative (E⁻) populations, which were prepared from blood of healthyhuman donors as described by Mendes (J. Immunol. 111:860, 1973).Detection of mouse hybridoma antibodies binding to these cells wasdetermined by radioimmunoassay and/or indirect immunofluorescence. Inthe first method, the cells were initially reacted with 100 μl ofaffinity-purified ¹²⁵ I goat-anti-mouse IgG (10⁶ cpm/μg; 500 μg/μl).(Details of iodination of goat-anti-mouse were described by Kung, etal., J. Biol. Chem. 251(8):2399, 1976). Alternatively, cells incubatedwith culture supernatants were stained with a fluorescinatedgoat-anti-mouse IgG (G/M FITC) (Meloy Laboratories, Springfield, VA;F/p=2.5) and the fluorescent antibody-coated cells were subsequentlyanalyzed on the Cytofluorograf FC200/4800A (Ortho Instruments, Westwood,MA) as described in Example III. Hybridoma cultures containingantibodies reacting specifically with E⁺ lymphocytes (T cells) wereselected and cloned. Subsequently, the clones were transferredintraperitoneally by injecting 1×10.sup. 7 cells of a given clone (0.2ml volume) into Balb/cJ mice primed with2,6,10,14-tetramethylpentadecane, sold by Aldrich Chemical Company underthe name Pristine. The malignant ascites from these mice were then usedto characterize lymphocytes as described below in Example II. thesubject hybrid antibody OKT1 was demonstrated by standard techniques tobe of IgG₁ subclass.

EXAMPLE II Characterization of OKT1 Reactivity A. Isolation ofLymphocyte Populations

Human peripheral blood mononuclear cells were isolated from healthyvolunteer donors (ages 15-40) by Ficoll-Hypaque density gradientcentrifugation (Pharmacia Fine Chemicals, Piscataway, NJ) following thetechnique of Boyum, Scand. J. Clin. Lab. Invest. 21 (Suppl. 97): 77,1968. Unfractionated mononuclear cells were separated into surface Ig⁺(B) and Ig⁻ (T plus Null) populations by Sephadex G-200 anti-F(ab')₂column chromatography as previously described by Chess, et al., J.Immunol. 113:1113 (1974). T cells were recovered by E rosetting the Ig⁻population with 5% sheep erythrocytes (Microbiological Associates,Bethesda, MD). The rosetted mixture was layered over Ficoll-Hypaque andthe recovered E⁺ pellet treated with 0.155M NH₄ Cl (10 ml per 10⁸cells). The T cell population so obtained was <2% EAC rosette positiveand >95% rosette positive as determined by standard methods. Inaddition, the non-rosetting Ig⁻ (Null cell) population was harvestedfrom the Ficoll interface. This latter population was <5% E⁺ and ≦2%Ig⁺. The surface Ig⁺ (B) population was obtained from the Sephadex G-200column following elution with normal human gamma globulin as previouslydescribed. This population was >95% surface Ig⁺ and <5% E⁺.

Normal human macrophages were obtained from the mononuclear populationby adherence to polystyrene. Thus, mononuclear cells were resuspended infinal culture media (RPMI 1640, 2.5 mM HEPES[4-(2-hydroxyethyl)-1-piperazinepropane sulfonic acid] buffer, 0.5%sodium bicarbonate, 200 mM L-glutamine, and 1% penicillin-streptomycin,supplemented with 20% heat-inactivated human AB serum) at aconcentration of 2×10⁶ cells and incubated in plastic petri dishes(100×20 mm) (Falcon Tissue Culture Dish; Falcon, Oxnard, CA) at 37° C.overnight. After extensive washing to remove non-adherent cells, theadherent population was detached by brisk washing with cold serum-freemedium containing 2.5 mM EDTA and occasional scraping with the rubbertip of a disposable syringe plunger. Greater than 85% of the cellpopulation was capable of ingesting latex particles and had morphologiccharacteristics of monocytes by Wright-Giemsa staining.

B. Normal Thymus

Normal human thymus gland was obtained from patients aged two months to14 years undergoing corrective cardiac surgery. Freshly obtainedportions of the thymus gland were immediately placed in 5% fetal calfserum in medium 199 (Gibco), finely minced with forceps and scissors,and subsequently made into single cell suspensions by being pressedthrough wire mesh. The cells were next layered over Ficoll-Hypaque andspun and washed as previously described in section A above. Thethymocytes so obtained were >95% viable and ≧90% E rosette positive.

C. Cell Lines

Epstein-Barr Virus (EBV) transformed B cell lines from four normalindividuals were prepared as previously described. T cell lines CEM,HSB-2, and HJD-1 were provided by Dr. H. Lazarus, Sidney Farber CancerInstitute, Boston, MA.

D. T Acute Lymphoblastic Leukemia (T-ALL) Cells and T Chronic LymphaticLeukemia (T-CLL) Cells

Leukemia cells were obtained by 12 patients with T-ALL. Theseindividuals' cells had previously been determined to be of T celllineage by their spontaneous rosette formation with sheep erythrocytes(>20% E⁺) and reactivity with T cell specific hetero-antisera, anti-HTL(anti-B.K.) and A99, as previously described by Schlossman, et al.,Proc. Nat. Acad. Sci. 73:1288 (1976). Tumor cells from three individualswere reactive (TH₂ ⁺) with rabbit and/or equine anti-TH₂ while cellsfrom the remaining nine were non-reactive (TH₂ ⁻). Leukemic cells fromtwo patients with TH₂ ⁻ T-CLL were also utilized. Both acute and chronicT cell leukemia cells were cryopreserved in -196° C. vapor phase liquidnitrogen in 10% dimethylsulfoxide and 20% AB human serum until the timeof surface characterization. The tumor populations analyzed were >90%blasts by Wright-Giemsa morphology in all instances.

EXAMPLE III Cytofluorographic Analysis and Cell Separation

Cytofluorographic analysis of all cell populations was performed byindirect immunofluorescence with fluorescein-conjugated goat-anti-mouseIgG (G/M FITC) (Meloy Laboratories) on a Cytofluorograf FC200/4800A(Ortho Instruments). In brief, 1-2×10⁶ cells were treated with 0.15 mlOKT1 at a 1:1000 dilution, incubated at 4° C. for 30 minutes, and washedtwice. The cells were then reacted with 0.15 ml of a 1:40 dilution G/MFITC at 4° C. for 30 minutes, centrifuged, and washed three times. Thesecells were then analyzed on the Cytofluorograf and the intensity offluoresence per cell recorded on a pulse height analyzer. A similarpattern of reactivity was observed at a dilution of 1:30,000, butfurther dilution caused loss of reactivity. Background staining wasobtained by substituting a 0.15 ml aliquot of 1:1000 ascites from aBalb/cJ mouse intraperitoneally immunized with a non-producing hybridclone.

In experiments designed to separate OKT1⁺ and OKT1⁻ cells, 100×10⁶unfractionated mononuclear cells or thymocytes were labeled with 4 ml ofa 1:1000 dilution of OKT1 and developed with G/M FITC. An identicalstaining approach was utilized to prepare human T cells isolated as inExample IIA above. Utilizing a fluorescence activated cell sorter(FACS-I) (Becton-Dickinson, Mountain View, CA), lymphocytes wereseparated into OKT1⁺ and OKT1⁻ populations and/or T cells werefractionated into weakly reactive OKT1⁺ T cells (lower 20% offluorescence) and strongly reactive OKT1⁺ T cells (upper 20%fluorescence). Post sort viability was >95% by Trypan blue exclusion inall instances. Purity of all separated populations was ≧95%.

EXAMPLE IV Functional Studies

The mitogenic response of the unseparated and FACS fractionated lymphoidcells was tested in microculture to optimal doses of Concanavalin A (ConA) (Calbiochem, La Jolla, CA) and phytohemagglutinin (PHA) as previouslydescribed by Chess, et al. Alloantigen proliferative response wasmeasured concurrently for these same populations using mitomycin treatedLaz 156, an EBV transformed human B lymphoblastoid cell line obtainedfrom Dr. H. Lazarus, as a stimulus. Proliferation to tetnus toxoid(Massachusetts Department of Public Health Biological Laboratories,Boston, MA) was tested as previously described by Evans, et al., (J.Immunol. 129: 1423, 1978), using a 10 μg/ml final concentration. Fivepercent macrophages obtained in the manner described above were added toall populations at the initiation of in vitro cultures. Mitogenstimulated cultures were pulsed after four days with 0.2 μCi oftritiated thymidine (1.9 Ci/mM specific activity; Schwartz-Mann Divisionof Becton-Dickinson, Orangeburg, NY) and harvested 18 hours later on aMASH II appartus (Microbiological Associates, Bethesda, MD). Tritiatedthymidine incorporation was measured in a Packard Scintillation Counter(Packard Instrument Company, Downer's Grove, IL). Background tritiatedthymidine incorporation was obtained by substituting medium for mitogen.Tetnus toxoid- and alloantigen-stimulated cultures were pulsed afterfive days with tritiated thymidine for 18 hours, harvested, and countedas described above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the fluorescence pattern obtained on the Cytofluorografafter reacting the cell populations shown with OKT1 at a 1:1000 dilutionand G/M FITC.

FIG. 2 shows the fluorescence pattern obtained on the Cytofluorografafter reacting human thymocytes with OKT1 and G/M FITC.

FIG. 3 shows the fluorescence pattern obtained on the Cytofluorografafter reacting leukemic cells from both acute lymphoblastic leukemia andchronic lymphoblastic leukemia patients with OKT1 and G/M FITC.

FIG. 4 shows the fluorescence pattern obtained on the Cytofluorografafter reacting human T cell lines with OKT1 and G/M FITC.

FIG. 5 shows the fluorescence pattern obtained on the Cytofluorografafter reacting the B cell lymphoblastoid line Laz 007 with OKT1 and G/MFITC.

The production of the hybridoma and the production and characterizationof the resulting monoclonal antibody were conducted as described in theabove Examples. Although large quantities of the subject antibody wereprepared by injecting the subject hybridoma intraperitoneally into miceand harvesting the malignant ascites, it is clearly contemplated thatthe hybridoma could be cultured in vitro by techniques well-known in theart and the antibody removed from the supernatant.

A sample of the subject hybridoma was deposited at the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, MD, 20852 on Mar.13, 1979, and has been assigned the ATCC number CRL 8000.

As shown in FIG. 1, the entire human peripheral blood T cell populationof a given normal individual is reactive with OKT1, whereas the entire Bcell, null cell, and macrophage populations isolated from the sameindividual are unreactive with OKT1. Similar results were obtained onpopulations of lymphocytes from fifteen other normal individuals. Themonoclonal antibody is thus characterized in that it is reactive with anantigen contained on the surface of essentially all normal humanperipheral T cells, while being unreactive with any antigens on thesurface of the other three cell types shown in FIG. 1. This differentialreactivity is one test by which the subject antibody OKT1 may bedetected and distinguished from other antibodies.

As shown in FIG. 2, the vast majority of normal human thymocytes from asix-month old infant are completely unreactive with OKT1, while about 5to 10 percent of the thymocytes are reactive. The implication of thisfinding is that, during the differentiation process by which stem cellsare converted into mature T cells, the thymocytes acquire at some stagethe same surface antigen found on T cells, which is reactive with OKT1.It is believed that these thymocytes are in the later stages ofdifferentiation just prior to emergence from the thymus into thebloodstream. Similar results (5-10% reactivity) were obtained using sixadditional thymus specimens from normal individuals two months to 19years of age. The pattern of reactivity in FIG. 2 provides a secondmethod of detecting the subject antibody OKT1 and distinguishing it fromother antibodies.

A diagnostic use for the subject antibody is illustrated by FIG. 3, inwhich it is shown that leukemic cells from T acute lymphoblasticleukemia (T-ALL) patients were nonreactive with OKT1, whereas leukemiccells from T chronic lymphoblastic leukemia (T-CLL) patients werereactive with OKT1. The subject antibody therefore provides a method fordistinguishing between these two forms of leukemia. Since it isdifficult to distinguish between certain stages of T-ALL and T-CLL andsince both the prognosis and the treatment regimen differ substantiallybetween these two forms of leukemia, it can be seen that astraightforward method for distinguishing between the two provided byuse of the subject antibody is a significant advance.

A further characterization of the subject antibody OKT1 is shown by thereactivity to various human T cell lines illustrated in FIG. 4. As canbe seen, the reactivity of the subject antigen to human T cell lines washeterogeneous, being strong for the line HJD-1, moderate for the lineCEM, and nonexistent for the line HSB-2. This differential reactivity ofOKT1 to various readily-available human T cell lines provides yetanother method of characterizing and describing the subject antibody.

FIG. 5 illustrates the lack of reaction of OKT1 with the human B cellline Laz 007. An identical pattern was obtained on the other EBVtransformed B cell lines tested. This further supports the lack ofreactivity of OKT1 with B cells obtained from the peripheral blood of anormal human population and provides yet another method forcharacterizing and distinguishing the subject antibody OKT1.

Functional studies were conducted on lymphoid populations which had beenseparated on a fluorescence activated cell separator (FACS). The resultsof these studies are shown in Tables I through III below and providefurther support for the previously-described characterization of thesubject monoclonal antibody.

As shown in Table I, essentially all of the responsiveness to PHA, ConA, soluble antigens, and alloantigen in mixed lymphocyte culture (MLC)resides in the population of cells responsive to OKT1. The populationwhich was unreactive to OKT1 appeared to cause none of these T cellfunctions, the slight response being accounted for by possiblecontamination with OKT1⁺ cells. These functional studies are a furtherillustration that the antigen to which OKT1 is reacting resides only onT cells, since the population which is so reactive exhibits T cellfunctions, while the population which is not so reactive exhibits noneof these functions. Table II illustrates that no functional differencesin mitogen or alloantigen response exist between the strongly OKT1reactive and weakly OKT1 reactive T cells separated on FACS. Bothpopulations proliferated equally well and in a manner identical to theunfractionated T cell population. Table III suggests that the surfaceantigen with which OKT1 reacts is present only on mature thymocytes,since the activity of the entire range of thymocytes in the MLC assay isdue almost entirely to that portion of the thymocyte population which isreactive with OKT1. Table III also shows the functional differencebetween OKT1⁺ lymphocytes and OKT1⁺ peripheral T cells, since the formerlack mitogen responsiveness.

According to the present invention there are provided a hybridomacapable of producing antibody against an antigen found on essentiallyall normal human T cells, a method for producing this hybridoma,monoclonal antibody against an antigen found on essentially all human Tcells, methods for producing the antibody, and methods for treatment ordiagnosis of disease employing this antibody.

Although only a single hybridoma producing a single monoclonal antibodyagainst human T cell antigen is described, it is contemplated that thepresent invention encompasses all monoclonal antibodies exhibiting thecharacteristics described herein. It was determined that the subjectantibody OKT1 belongs to the subclass IgG₁, which is one of foursubclasses of murine IgG. These subclasses of immune globulin G differfrom one another in the so-called "fixed" regions, although an antibodyto a specific antigen will have a so-called "variable" region which isfunctionally identical regardless of which subclass of immune globulin Git belongs to. That is, a monoclonal antibody exhibiting thecharacteristic described herein may be of subclass IgG₁, IgG₂ a, IgG₂ b,or IgG₃, or of classes IgM, IgA, or other known Ig classes. Thedifferences among these classes or subclasses will not affect theselectivity of the reaction pattern of the antibody, but may affect thefurther reaction of the antibody with other materials, such as (forexample) complement or anti-mouse antibodies. Although the subjectantibody is specifically IgG₁, it is contemplated that antibodies havingthe patterns of reactivity illustrated herein are included within thesubject invention regardless of the immune globulin class or subclass towhich they belong.

Further included within the subject invention are methods for preparingthe monoclonal antibodies described above employing the hybridomatechnique illustrated herein. Although only one example of a hybridomais given herein, it is contemplated that one skilled in the art couldfollow the immunization, fusion, and selection methods provided hereinand obtain other hybridomas capable of producing antibodies having thereactivity characteristics described herein. Since the individualhybridoma produced from a known mouse myeloma cell line and spleen cellsfrom a known species of mouse cannot be further identified except byreference to the antibody produced by the hybridoma, it is contemplatedthat all hybridomas producing antibody having the reactivitycharacteristics described above are included within the subjectinvention, as are methods for making this antibody employing thehybridoma.

Further aspects of the invention are methods of treatment or diagnosisof disease employing the monoclonal antibody OKT1 or any othermonoclonal antibody exhibiting the pattern of reactivity providedherein. As discussed above, the subject antibody allows discriminationbetween T cell chronic lymphoblastic leukemia and T cell acutelymphoblastic leukemia and allows treatment of patients undergoing organtransplants to reduce or eliminate the rejection of these transplants.

                                      TABLE I                                     __________________________________________________________________________    FUNCTIONAL COMPARISON OF FACS-SEPARATED OKTl.sup.+  AND OKTl.sup.-            PERIPHERAL LYMPHOCYTES                                                                       Whole Mononuclear                                              Proliferative                                                                        Whole   OKTl + G/M FITC                                                Stimulus                                                                             Mononuclear                                                                           Treated    OKTl.sup.+                                                                            OKTl.sup.-                                  __________________________________________________________________________    Con A  146,032 ± 1,556                                                                    137,229 ± 3,600                                                                       133,557 ± 6,088                                                                    9,454 ± 1,080                            PHA     32,001 ± 2,659                                                                     36,326 ± 3,311                                                                        29,877 ± 1,043                                                                    8,058 ± 869                              MLC    122,958 ± 2,315                                                                    136,141 ± 1,056                                                                       148,235 ± 2,666                                                                    8,125 ± 1,033                            Tetanus                                                                               25,821 ± 4,132                                                                     28,756 ± 1,526                                                                       30,184 ± 563                                                                       2,124 ± 436                              Toxoid                                                                        Media   482 ± 16                                                                            734 ± 65                                                                              533 ± 87                                                                         757 ± 108                                Control                                                                       __________________________________________________________________________

                                      TABLE II                                    __________________________________________________________________________    FUNCTIONAL COMPARISON OF T CELLS STRONGLY REACTIVE AND WEAKLY                 REACTIVE WITH OKTl                                                                            Unfractionated                                                                T cells    T cells strongly                                                                       T cells weakly                            Proliferative                                                                        Unfractionated                                                                         OKTl + G/M FITC                                                                          reactive reactive                                  Stimulus                                                                             T cells  Treated    with OKTl                                                                              with OKTl                                 __________________________________________________________________________    EXPERIMENT #1                                                                 Con A  59,499 ± 9,699                                                                      56,248 ± 3,057                                                                        64,656 ± 6,076                                                                      54,478 ± 5,173                         PHA    116,062 ± 5,910                                                                     106,412 ± 5,348                                                                       112,246 ± 3,716                                                                     90,857 ± 5,500                         MLC    95,261 ± 4,663                                                                      107,365 ± 12,001                                                                      119,605 ± 5,333                                                                     100,650 ± 8,215                        Media  365 ± 22                                                                            399 ± 46                                                                              488 ± 23                                                                            402 ± 57                               EXPERIMENT #2                                                                 Con A  88,603 ± 2,133                                                                      104,241 ± 1,951                                                                       99,617 ± 7,213                                                                      117,672 ± 12,315                       PHA    79,235 ± 2,615                                                                      65,803 ± 6,163                                                                        73,108 ± 2,226                                                                      67,159 ± 6,316                         MLC    39,096 ± 5,776                                                                      35,929 ± 2,102                                                                        55,009 ± 8,333                                                                      42,165 ±  4,559                        Media  157 ± 28                                                                            292 ± 6 322 ± 33                                                                            345 ± 25                               __________________________________________________________________________

                                      TABLE III                                   __________________________________________________________________________    FUNCTIONAL PROPERTIES OF HUMAN THYMOCYTE POPULATIONS                          Proliferative                                                                        Unfractionated                                                                        Unfractionated Thymocytes                                                                    OKTl.sup.+                                                                           OKTl.sup.-                               Stimulus                                                                             Thymocytes                                                                            OKTl + G/M FITC Treated                                                                      Thymocytes                                                                           Thymocytes                               __________________________________________________________________________    EXPERIMENT #1                                                                 MLC    7,085 ± 901                                                                        6,224 ± 823 6,556 ± 987                                                                       244 ± 10                              Con A   88 ± 10                                                                           94 ± 2      38 ± 2                                                                            36 ± 3                                PHA    55 ± 6                                                                             78 ± 10     22 ± 2                                                                            51 ± 4                                Media  40 ± 5                                                                             46 ± 10     10 ± 2                                                                             65 ± 11                              EXPERIMENT #2                                                                 MLC    3,815 ± 772                                                                        4,778 ± 623 5,727 ± 239                                                                       425 ± 81                              Con A  46 ± 8                                                                             47 ± 10     100 ± 22                                                                           67 ± 32                              PHA    66 ± 4                                                                             60 ± 4      142 ± 4                                                                           22 ± 4                                Media   80 ± 15                                                                           67 ±  12     80 ± 18                                                                          200 ± 16                              __________________________________________________________________________

What is claimed is:
 1. An IgG monoclonal-antibody-producing hybridomaformed by fusion of cells from a mouse myeloma line and spleen cellsfrom a mouse previously immunized with human T cells, which antibody:(a)reacts with essentially all normal human peripheral T cells but not withnormal human peripheral B cells, null cells or macrophages; (b) reactswith from about 5% to about 10% of normal human thymocytes; (c) reactswith leukemic cells from humans with T cell chronic lymphoblasticleukemia but does not react with leukemic cells from humans with T cellacute lymphoblastic leukemia; (d) exhibits a pattern of reactivity withthe human T cell lines HJD-1, CEM, and HSB-2 shown in FIG. 4; and (e)does not react with Epstein-Barr virus-transformed human B cell lines.2. The hybridoma of claim 1 wherein the antibody produced thereby is ofsubclass IgG₁.
 3. The hybridoma of claim 1 which is formed by fusion ofP3X63AgU1 myeloma cells and spleen cells from a Balb/cJ mouse previouslyimmunized with E rosette purified human T cells.
 4. A hybridomaaccording to claim 1 having the identifying characteristics of ATCCnumber CRL
 8000. 5. A hybridoma formed by fusion of cells from a mousemyeloma line and spleen cells from a mouse previously immunized withhuman T cells, which hybridoma is capable of producing a monoclonalantibody which reacts with essentially all normal human peripheral Tcells but not with normal human peripheral human B cells, null cells, ormacrophages.
 6. A hybridoma capable of producing mouse monoclonalantibody that reacts with essentially all normal human peripheral Tcells but not with normal human peripheral B cells, null cells, ormacrophages.
 7. A hybridoma capable of producing mouse monoclonalantibody that reacts with essentially all normal human peripheral Tcells.